Parafilm

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

Parafilm is a flexible, self-sealing laboratory film made of a blend of polyolefins. It is designed to create a barrier against air, moisture, and light, making it useful for sealing and covering a variety of laboratory containers and equipment.

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20 protocols using parafilm

VOC Analysis of Drug-Free Urine

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UTAK drug-free normal urine, Parafilm, pH paper, and polypropylene cups for urine collection were purchased from ThermoFisher Scientific (Waltham, MA, USA). Sodium hydroxide (50% wt. in solution, nitrogen flushed extra pure) and sodium chloride (99.85% pure) were also purchased from ThermoFisher Scientific. Guanidine hydrochloride (GHCl; pH = 8.5) was purchased from Sigma Aldrich (St. Louis, MO, USA). SPME fibers coated with DVB/CAR/PDMS (two centimeters in length) were purchased from Supelco (Bellefonte, PA, USA). SPME arrows of a similar chemical composition (DVB/Carbon Wide Range/PDMS) were obtained from Restek (Bellefonte, PA, USA). Headspace vials (10 mL) with screw-on caps were purchased from Restek or Agilent (Santa Clara, CA, USA). An Agilent 7890A GC system coupled to an Agilent 7200 MS quadrupole time-of-flight (QTOF) equipped with a PAL autosampling system (CTC Analytics, Zwingen, Switzerland) was used to incubate, extract, and analyze the VOCs. The GC column utilized for VOC separation was a Restek Rxi-5ms column of 30 m in length, a 0.25 mm internal diameter, and a 0.25 μm film thickness.

Woollam M., Siegel A.P., Munshi A., Liu S., Tholpady S., Gardner T., Li B.Y., Yokota H, & Agarwal M. (2023). Canine-Inspired Chemometric Analysis of Volatile Organic Compounds in Urine Headspace to Distinguish Prostate Cancer in Mice and Men. Cancers, 15(4), 1352.

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Immunofluorescence Staining of BxPC-3 Cells

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BxPC-3 cells, cat. no. CRL-1687 (ATCC, Manassas, VA)

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Cover Glasses, round, 18 mm diameter, cat. no. 12-548A (ThermoFisher Scientific, Waltham, MA).

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Sterile 12-well multi-well plates, cat. no. 712001 (Bioland Scientific, Paramount, CA)

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Forceps, fine tip, 5 in., cat. no. 3120019 (ThermoFisher Scientific, Waltham, MA).

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Glass slides, cat. no. 12550003 (ThermoFisher Scientific, Waltham, MA).

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Parafilm, cat. no.1337410 (ThermoFisher Scientific, Waltham, MA).

Baik M., French B., Chen Y.C., Byers J.T., Chen K.T., French S.W, & Díaz B. (2019). Identification of invadopodia by TKS5 staining in human cancer lines and patient tumor samples. MethodsX, 6, 718-726.

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Zika Virus Vector Competence Assay

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Adult female mosquitoes were challenged with a ZIKV-containing blood-meal at 5 days post-eclosion. ZIKV I-44 strain (Genbank: KX856011) was used for challenges, isolated from mosquitoes from Mexico in 2016 [1 (link),24 (link)]. Prior to its use in vector competence studies, the virus had been serially passaged four times in Vero cells. ZIKV was added to 90% confluent Vero cells (ATCC: CCL-81) at multiplicity of infection (MOI) 0.01 for 96–120 hours or until 70% cytopathic effect (CPE) was observed. Vero cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 7% fetal bovine serum (FBS). Virus-containing supernatant was harvested and used immediately for artificial feedings in a 1:1 ratio with defibrinated sheep blood (Colorado Serum Company, Denver, CO, USA) supplemented with 10mM ATP to stimulate feeding. For each mosquito carton, the blood-meal was supplied through a parafilm (Thermo Fisher Scientific, Waltham, MA, USA) membrane stretched over a glass feeder, with the glass feeder heated by a water-jacket to 37°C. Mosquitoes were provided the infectious blood-meal for up to 1 hour and anaesthetised on ice for selection of engorged females, which were then kept in cardboard cartons in a humidified chamber at 28°C and 80% humidity until sampled.

Herd C.S., Grant D.G., Lin J, & Franz A.W. (2021). Starvation at the larval stage increases the vector competence of Aedes aegypti females for Zika virus. PLoS Neglected Tropical Diseases, 15(11), e0010003.

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Measuring Radiation-Induced Senescence in Fanca Cells

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129/Sv Fanca^+/+ and 129/Sv Fanca^−/− bone marrow stromal cell lines were plated in duplicate in six-well plates, incubated overnight to allow attachment, then irradiated to various doses using a Shephard Mark I ¹³⁷Cs gamma-ray source. Cells were kept at sub-confluence and grown in DMEM supplemented with 10% FBS, 1% antibiotic-antimycotic solution and 1% l-glutamine. Cells were incubated at 37°C in 5% CO₂. Drugs were added to media 1 h prior to incubation and supplemented in media continuously afterwards.
At indicated time points, chromogenic beta-galactosidase assay (Cell Signaling Technology®, Inc., Danvers, MA) was performed according to the manufacturer’s protocol. Briefly, cells were washed with phosphate buffered saline (PBS) then incubated 4 min with 1× fixative solution. Cells were washed with PBS twice, then beta-galactosidase solution was added. Beta-galactosidase solution was prepared fresh for each assay and titrated to pH 6.0 (28 (link)). Plates were wrapped with Parafilm® (Thermo Fisher Scientific) and incubated overnight at 37°C in 0% CO₂. Cells were washed with PBS twice, then with methanol once. Samples were plated in duplicate. The percentage of positive blue cells was counted using light microscopy at 20× magnification, ensuring a minimum of 500 cells counted per sample (28 (link)).

Sivananthan A., Shields D., Fisher R., Hou W., Zhang X., Franicola D., Epperly M.W., Wipf P, & Greenberger J.S. (2018). Continuous One Year Oral Administration of the Radiation Mitigator, MMS350, after Total-Body Irradiation, Restores Bone Marrow Stromal Cell Proliferative Capacity and Reduces Senescence in Fanconi Anemia (Fanca−/−) Mice. Radiation research, 191(2), 139-153.

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Tumor Development on Chick Chorioallantoic Membrane

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Tumor development on CAM was performed as previously described [29 (link)]. Briefly, fertilized eggs (animal facility of the University of Geneva, Geneva, Switzerland) were incubated at 38 °C with 80% relative humidity and periodic rotation. On egg development day (EDD) 4, the eggs were drilled at their narrow apex, and the hole was closed with adhesive tape. The eggs were then incubated at 38 °C with 80% relative humidity without rotation. On EDD8 the hole in the eggshell was enlarged to allow access to the CAM. After gently scratching the membrane with a needle tip, SKOV3-Red, SKOV3-Green, or SKOV3-M cells suspension (2 × 10⁶ cells in 30 μL of geltrex, Thermo Scientific) was inoculated, and the hole was hermetically covered with Parafilm®. Eggs were returned to the incubator to allow tumor growth. Tumor growth was then monitored at EDD10.5 and 13.5 using a Wild Heerbrugg M3Z microscope at 10x magnification with a Lumenera INFINITY2-1 CDD camera with Infinity Capture Software.

Yart L., Bastida-Ruiz D., Allard M., Dietrich P.Y., Petignat P, & Cohen M. (2022). Linking unfolded protein response to ovarian cancer cell fusion. BMC Cancer, 22, 622.

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Polymer-based Excipient Formulation Development

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Sodium polyacrylate was purchased from Magnacol Ltd. (Newtown, Wales). Magnesium stearate, parafilm, and double-sided polytetrafluoroethylene (PTFE) adhesive were purchased from Fischer Scientific (Loughborough, UK). The polyvinyl alcohol–polyethylene glycol graft copolymer (Kollicoat^® IR, denoted as KIR) was provided by BASF GmbH (Ludwigshafen, Germany). Albumin, α-lactose monohydrate, gelatin type A from porcine (bloom strength 90), λ-carrageenan from red seaweed (denoted as λ-C), hydroxypropyl methylcellulose (HPMC; grade 2910 and average M_w of 8000 Da), and polyvinyl alcohol (referred to as PVA; average M_w 31,000 of Da and 98–99% hydrolyzed) were all purchased from Sigma-Aldrich, (Poole, UK). Potassium phosphate, sodium fluoride, and calcium chloride were purchased from RM Marketing (Essex, UK). Aerosil^® 200 was from Evonik GmbH, (Wesseling, Germany), and Avicel^® PH102 was from DuPont (Delaware, USA). Propyl-methacrylate-polyvinyl acetate phthalate (PMPP) clear resin was obtained from Formlabs GmbH, Germany. Opadry^® EZ (referred to as OEZ) formulations were obtained from Colorcon (Colorcon Ltd., Dartford, UK).

Ershad A.L., Rajabi-Siahboomi A., Missaghi S., Kirby D, & Mohammed A.R. (2021). Multi-Analytical Framework to Assess the In Vitro Swallowability of Solid Oral Dosage Forms Targeting Patient Acceptability and Adherence. Pharmaceutics, 13(3), 411.

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Intramuscular Injection of Cryopreserved MPC

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Cryopreserved MPC were freshly thawed, washed once with 1X PBS, and centrifuged at 400×g for 10 min followed by resuspension in 1X PBS to reach a final concentration of 40 × 10⁶ cells/mL. Twenty-five microliters of the MPC suspension (containing 1 × 10⁶ cells) were mixed on a Parafilm with 5 μL FluoSpheres® polystyrene beads (15 μm, yellow-green or scarlet, Thermo-Fisher Scientific, USA), in order to track MPC within the tibialis anterior muscle during follow-up (Supplementary Fig. 2C). MPC were injected intramuscularly using a custom-made injector (Innerbichler GmbH, Austria) containing 4 needles (30G) mounted on a 1 mL syringe (Braun, Germany).

Messner F., Thurner M., Müller J., Blumer M., Hofmann J., Marksteiner R., Couillard-Despres S., Troppmair J., Öfner D., Schneeberger S, & Hautz T. (2021). Myogenic progenitor cell transplantation for muscle regeneration following hindlimb ischemia and reperfusion. Stem Cell Research & Therapy, 12, 146.

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Mechanical Stimulation of Skin Equivalents

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A novel mechanical system was constructed in-house to provide both mechanical movement to fixation pin as well as provide a suitable environment for culturing the skin models (Figure 1). A linear actuator L16-R (Actuonix Motion Devices, Canada) was employed to generate the linear displacement of pins and an Arduino ATMEGA 2560 microcontroller (Arduino™, Italy) was used to control the magnitude and frequency of the linear displacement. The skin models were grown on 24 mm Transwell® inserts, which were placed in a custom-designed aluminium well system and covered in Parafilm™ (Thermo Fisher Scientific, UK) to maintain sterility (Figure 2). The pins were implanted by pushing the pin through the HSE into a concentric hole at the base of the well. A stopcock at the base of the well allowed for the sampling of media for analysis or the addition of fresh medium to maintain an air-liquid interface.

McCall B., Rana K., Sugden K, & Junaid S. (2024). In-vitro external fixation pin-site model proof of concept: A novel approach to studying wound healing in transcutaneous implants. Proceedings of the Institution of Mechanical Engineers. Part H, Journal of Engineering in Medicine, 238(4), 403-411.

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Transformation of Chemically Competent E. coli

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50 μl ONE Shot TOP10 or ONE SHOT Stbl3 Chemically Competent E.coli (Thermo Fisher Scientific) were thawed on ice for each transformation. 5 μl plasmid DNA was added to the competent cells and mixed by flicking the tube before storing on ice for 30 min. Cells were then placed at 42°C for 30 seconds to enable incorporation of DNA and then placed on ice for 2 min. 250 μl of Super Optimal broth with Catabolite repression (SOC) medium at room temperature was added to each vial and placed in a 37°C orbital shaker for 2 h. After incubation, 20 μl and 200 μl of each transformation was placed on a pre-warmed agar plate containing the appropriate antibiotic and spread using ColiRoller Plating Beads (Millipore). Plates were wrapped in Parafilm (Thermo Fisher), inverted and placed in a 37°C incubator overnight. Plates were checked the next day for bacterial colonies and placed at 4°C until required.

Taylor C., Mannion D., Mirand a F., Karaminejadranjbar M., Herrero-Gonzalez S., Hellner K., Zheng Y., Bartholomeusz G., Bast RC J.r, & Ahmed A.A. (2017). Loss of PFKFB4 induces cell death in mitotically arrested ovarian cancer cells. Oncotarget, 8(11), 17960-17980.

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Sub-lethal Thermal Stress Responses

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SMMC-7721, HepG2, and MHCC97-H cells were sub-lethally heated (50 °C) for 10 min. Heat treatments were carried out by sealing the culture bottle with Parafilm, and submerging the plates in a water bath (HH · W21 · 600S, Shanghai, China) set at 50 °C and returned to the incubation chamber (Series II Water Jacket, Thermo-Scientific, Waltham, MA) for 24 h at 37 °C. Three independent experiments were performed.

Deng Q., Chen S., Fu C., Jiang J., Zou M., Tan Y., Wang X., Xia F., Feng K., Ma K, & Bie P. (2017). Long noncoding RNA expression profiles in sub-lethal heat-treated hepatoma carcinoma cells. World Journal of Surgical Oncology, 15, 136.

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